TITLE CHANGE: FOLLOWING SINGLE MESSENGER RNA MOLECULES FROM BIRTH TO DEATH IN YEAST Over the last funding period, we have developed real-time single molecule technology that provides unparalled insight into fundamental processes of gene expression: transcription, nuclear pore export, mRNA localization and decay. The methodology permits the detection and description of kinetic events occurring at a single gene, or with a single mRNA. It has become apparent that this technology can be further extended; for instance into additional areas of mRNA metabolism or into a finer temporal and spatial regulation. This is made possible by the development of two stem-loop aptamers derived from the phages MS2 and PP7 that can be inserted into any mRNA of interest and bound to two differently colored coat proteins. These different colors can be used to mark RNA intra- or intermolecular. For instance the elongation rates of individual polymerases can be measured using these two markers, the orientation of passage of mRNAs through the nuclear pore, the translational frequency of a single mRNA, or the moment it decays. Because this single molecule technology is done in single cells, it will provide us with range of variance among cells, providing an understanding of the elasticity and tolerances in the mechanism. Because of our focus has now changed to single molecule analysis, we have changed the title to: FOLLOWING SINGLE MESSENGER RNA MOLECULES FROM BIRTH TO DEATH to reflect this change in emphasis. PUBLIC HEALTH RELEVANCE: Project Narrative: This work details the development of innovative technology that uses novel microscopy to observe single RNA molecules in living cells. This makes our observations hundreds of times more sensitive that current methods and provides new insights into how cells work. Armed with this new information, we will be able to apply these methods to detect the likely points of malfunction and design remedies